The interacting-heads motif (IHM) is a configuration of myosin heads in relaxed thick filaments of muscle, in which ATP turnover and actin binding are inhibited by the interaction of each head with the other. In a dramatic development in the field, this motif has become recognized as a fundamental feature of normal muscle relaxation and contraction, through its regulation of thick filament activity. In addition to its role in thick filaments, the IHM also underlies the structure of single molecules of myosin II in almost all types of animal cell. In this monomeric form, the myosin tail folds up, forming a compact molecule, in which ATPase activity is again inhibited by similar head-head interactions. The IHM appears to play two key roles in the body. In thick filaments, it contributes to energy conservation in the relaxed state of muscle. As a monomer, it functions as a storage form of myosin whose compact form facilitates transport to its site of filament assembly. These critical new findings are the motivation for this application: our goal is to elucidate the structure of this fundamental regulatory motif in skeletal muscle thick filaments and single myosin molecules, thus illuminating how it functions. We will do this using state-of-the-art cryo-EM and 3D reconstruction techniques, studying selected model systems and integrating the information gained from each. In Aim 1 we will determine the 3D structure of the IHM in native thick filaments using novel cryo-EM technology that is currently revolutionizing structural biology. Using tarantula skeletal muscle filaments, the most stable species known, we will determine at better than 10 resolution the interactions between the two heads, and between the heads and the tail, that create the IHM. With help from the insights gained, we will determine the structure of frog skeletal thick filaments, the most stable vertebrate filament. And we will build on this information to determine the structure of the IHM in (less stable) mammalian thick filaments. In Aim 2, we will determine the 3D structure of the IHM in isolated myosin molecules, using three complementary systems: smooth muscle myosin as the most stable single molecule, which will provide the highest resolution; tarantula myosin as a direct link to the filament structure in Aim 1, aiding its interpretation; and mammalian myosin, which will reveal the structure in vertebrate skeletal muscle. We will also examine molecules in which putative head interaction sites have been mutated, to test their importance in formation of the IHM. In Aim 3, we will use single molecule EM to test the hypothesis that disease mutations in the head region of skeletal myosin impact the stability of the IHM. The IHM is now recognized as a fundamental motif of normal muscle function. Our proposal will elucidate its interactions, providing new insights into the structural basis of contraction and relaxation, and of the potential impact of disease mutations on these functions.